Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

BACKGROUND: Biomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts.

METHODS: To this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium.

RESULTS: The CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.

CONCLUSIONS: This work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.